A strip-type immunoassay for immunochromatography has become widely applicable as a simple in vitro diagnostic kit or a portable diagnostic device that makes use of specific reactivity of antibodies to detect a substance to be detected (e.g., antigens) in a sample liquid. A simple immunochromatographic testing instrument for testing the presence or absence of an infection caused by pathogens such as influenza virus and bacteria has attracted interest, and there has been ongoing research and development of such instruments.
Diagnosis of hemolytic streptococcus (hereinafter, also referred to as “streptococcus”) is performed using group specific polysaccharides as antigens. Known polysaccharide extraction methods include methods using enzymes or phages, and methods using hydrochloric acid or hypochlorous acid. The extraction method using nitrous acid is most common.
The extraction method using nitrous acid has merits in its high polysaccharide extraction efficiency, and the low price and easy handling of nitrous acid. A demerit is that a nitrous acid itself is an unstable compound that easily decomposes, and needs to be prepared for each use by mixing sodium nitrite and organic acids such as acetic acid prior to extraction. Such preparation of a nitrous acid puts a large burden on physicians and laboratory technicians when diagnosis needs to be made regularly. Another drawback is that, because of the mixing step, an error may occur in mixing the reagents, and the method may fail to properly and safely perform diagnosis.
In order to overcome these problems, there has been research and development of simple testing instruments that simplify the step of extracting polysaccharide antigens from streptococci.
For example, Patent Document 1 proposes a method for extracting polysaccharide antigens from organisms (particularly, Group A or B streptococci). The method simplifies the extraction with the use of a kit that combines a) a dry first absorbent material impregnated with a premeasured amount of nitrite, b) a dry second absorbent material impregnated with a premeasured amount of a neutralizing base and buffer, and c) a premeasured amount of an aqueous solution of an acid (see Patent Document 1).
Diagnosis of a streptococcal infection requires the complicated process of performing a polysaccharide antigen extraction step, followed by an antigen detection step in which the resulting solution is contacted to an immunoassay device. There accordingly remains the challenge of developing a method or a kit therefor for conveniently measuring and testing organisms in a sample with a polysaccharide antigen extraction step, and a marker measurement step.
For example, Patent Document 2 discloses an assay device and method for the detection of carbohydrate antigens characteristic of microbial/bacterial organisms such as the family Streptococcacae. This lateral flow assay device includes a substrate having a) sample receiving zone, b) an extraction zone (extraction reagent; an immobilized or absorbed, and dried acid or nitrite), c) a neutralizing agent (neutralizing buffer; TRIS), and d) a detection zone (capture/detection reagent) (see Patent Document 2).
A method and a kit therefor for simultaneously measuring and testing different organisms containing streptococci are also developed.
For example, Patent Document 3 discloses a method and a kit for measuring a plurality of different biological species in a sample, such as in a sample containing a first organism that is a gam-positive bacterium, for example, such as Group A, B, F, or G streptococeus, and enterococcus bacteria, and a second organism that is a virus or a gram-negative bacterium. The simultaneous detection of a plurality of analytes is enabled by a kit that includes, in one or more containers, a) a nitrous acid, or a dry form of an acid or a nitrite, b) a surfactant, c) a first binding reagent that binds to a first marker obtained from the first organism, and d) a second binding reagent that binds to a second marker obtained from the second organism. This achieves doubling of test efficiency and alleviation of pain caused to patients by the test (see Patent Document 3).
An assay device and method for the detection of carbohydrate antigens characteristic of microbial/bacterial organisms such as the family Streptococcacae is commercially available. For example, QuickVue DipStick Strep A (DS Pharma Biomedical), and Strep A TestPack-plus OBC (Sanwa Kagaku Kenkyusho) are known examples of immunochromatography reagents. Known examples of slidelatex agglutination reagents include A Strept AD “Seiken” (Denka Seiken).
In order to produce a positive result in common testing of an analyte, a commercially available immunochromatography reagent requires a streptococcus concentration of at least 1×106 CFU/mL, in the direct method. The test thus may identify a positive specimen as negative when the streptococcus concentration is below 1×105 CFU/mL. An immunochromatographic test agent using antibodies labeled with an insoluble support is typically less sensitive than EIA, and involves such problems that an unclear line is observed in a positive case, and a positive result is provided even in the absence of a substance to be detected (e.g., antigens) in a sample liquid (i.e., false positive result is caused).
These problems are addressed by methods that use sugars or water-soluble high molecular compounds in a developing solvent. For example, a membrane assay is proposed that uses antibody-conjugated color latex particles. The assay uses an immunoassay latex composition of pH 9.0 to 9.8 containing at least one agglutination preventing agent such as sugars (for example, monosaccharides, and oligosacchasides, and sugar alcohols of such sugars) and polyalcohol; proteins; and a basic buffer. The composition prevents natural agglutination of latex particles, and increase of specific gravity, viscosity, and osmotic pressure to achieve high sensitivity immunoassay (see Patent Document 4).
In addition, recently, concerning a particle immunochromatography assay of glycohemoglobin (blood hemoglogin with the bound sugar) used an a suitable index of diabetes diagnosis, particularly hemoglobin A1c (HbA1c) with the glycosylated N-terminal valine residue in the hemoglobin β chain, there is proposed a detection method that includes (A) treating a red blood cell-containing measurement sample with a surfactant to expose the N-terminal of hemoglobin β chain on protein surface, (B) contacting the resulting sample to a cyclic polysaccharide of water-insoluble form (for example, a cyclic polysaccharide is immobilized on a membrane or the like by chemically bonding, a cyclic polysaccharide itself forms a polymer, or a cyclic polysaccharide is kneaded in a porous resin), and (C) contacting the resulting sample to antibodies or the like that recognize the N-terminal of the particle-labeled hemoglobin. In this way, the antibodies are prevented from agglutinating each other and failing to develop on the membrane, and the method prevents measurement inaccuracy caused when the constituent cyclic oligosaccharide molecule or cyclic polysaccharide molecule of the cyclic polysaccharide does not dissolve in water and does not diffuse upon contacting water (see Patent Document 5).
Concerning a simple method of testing an analyte using membrane assay, there is proposed an analyte sample filtration method that enables preventing yielding a false positive result or clogging while maintaining sensitivity (see Patent Document 6).
However, the immunochromatography technique (also referred to as “particle immunochromatography technique”) using antibodies labeled with an insoluble support (e.g., gold colloidal particles, and color latex particles) still involves agglutination of the insoluble support, and, possibly, non-specific reactions, depending on the measurement sample, the measurement environment, and the measurement conditions. Problems such as slow development rate may thus still remain. There accordingly is a strong need for pursuit of a test agent having a fast development rate and that does not cause agglutination of insoluble support or non-specific reactions even when used in the particle immunochromatography technique with different measurement samples under different measurement environments and conditions.